Expression in Escherichia coli, Purification by affinity chromatography, and Characterization through Gel Electrophoresis of protein gbr-22
Make intro more relevant to this lab
Introduction:
Lab strains of Escherichia coli bacteria serve as a sufficient expression host in recombinant protein production. This is because a wide variety of variables can be tested in a short amount of time and it is an inexpensive process [1]. The production of a protein with affinity tags, such as histidine, aid in protein purification. A histidine tag is small in size, rarely alters protein characteristics, and can be removed using metal affinity chromatography [1]. The lower-affinity contaminants will compete with the excess of histidine-tagged protein to farther purify the protein. To prepare the protein for these processes, enzymes that degrade nucleic acids and cell-wall material, such as Benzonase and lysozyme, should be administered to the protein solution. Imidazole can then be added to the cell extraction buffer to remove loosely bound proteins without removing large segments of the recombinant protein. Then, in an additional buffer elution step containing a stronger imidazole concentration, the protein can be removed and collected in the flow through to prepare for characterization. In protein characterization the molecular weight, purity, and yield of the protein will be analyzed through Gel electrophoresis. Gel electrophoresis requires the use of agarose gel, buffer solution, and dye [2]. In Gel Electrophoresis an electrical charge is applied to separate fragments based on their molecular weight and positive or negative migration. As the positively charges dyes migrate towards the cathode, the gel containing these dyes are placed in the wells of the tank so that the dyes will migrate through the gel towards the cathode [2].
Through a series of experimental procedures the recombinant protein, gbr-22, will be overexpressed in the bacterial host, E.coli. Gbr-22 will then be isolated and purified through affinity chromatography. The samples collected during the protein purification process will be used in Gel Electrophoresis to determine the estimated molecular weight, purity, and overall yield of the gbr-22 protein. The gbr-22 protein production and purification process will have some contamination present that will result in a protein purity of about 70%. This will later be demonstrated through gel analysis.
Materials & Methods:
In the protein expression process, two transformation tubes were centrifuged containing 25 microliters each of BL21(DE3) competent bacterial cells from NEB (put NEB in a citation & state). 1-2 microliters of plasmid (pGEM-gbr22) was administered into a transformation tube containing DNA; the other tube is the control (specify that control means no DNA). Heat shock tubes in 42 C waterbath for 45 seconds. Add SOC media to the incubator for 30 min at 37 C at 250 rpm and spread onto a plate to store overnight. Select a colony from the protein expression and transfer the cells into the LB/amp media to be placed in the shaking incubator. Grow at 37 C and 200-350 rpm. Centrifuge (Which centrifuge did you use) 500 ul (Centrifuged 25 mL, 500ul sample was taken before) of the sample for 10 min at 4 C and 5000 rpm. Add 1xPBS to the pellet, vortex, and add the enzyme lysozyme and store in a -4 C freezer.
For protein purification add 2 ul Benzonase, centrifuge for 20 min at 14,000 rpm and 4 C then run through 0.22 um filter (Which brand of filter). Add .5 ml of Ni-NTA buffer mix to the protein solution. Using a Econo column collect the flow through, leaving some of the purple pellet in fluid. Flow through the Wash buffer, 10ml of 1x PBS and 20mM imidazole, and twice with Elution buffer, 10mL of 1x PBS with 250 mM imidazole. Samples of cell solution, fraction, flow through, wash, Elu1, and Elu2 will be collected for use in gel electrophoresis. Use the Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE)/UV-VIS to measure the elution absorbance at 280nm and maximal wavelength.
In Protein characterization, add loading buffer to the samples collected in protein purification and administer into the cleared cell walls. Set the tank at 200 V for 25 min. Wash the gel and apply imperial protein stain. Cover gel with saran wrap and dry at 75 C for 1.5 hours on vacuum mode. (What type of gel did you use)
Results:
Figure 1: Day 2, 2/28/13, BL21(DE3), No DNA, VDS
Figure 2: Day 2, 2/28/13, BL21(DE3), pGEM-gbr22, Bacteria colonies (~1,000+) represented in light purple, VDS
Figure 3: Day 2, 2/28/13, Fun Plate after overnight incubation at 37 C, colonies grown from shoe swabs (~100 colonies) represented in yellow blotches, VDS
Figure 4: Day 2, Evening, 2/28/13, (LB+Amp), BL21(DE3), pGEM-gbr22, after being left overnight in the shaking incubator at 37 C and 200-350 rpm, VDS
Figure 5: Day 3, 2/29/13, (LB+Amp), BL21(DE3), pGEM-gbr22, purple cell pellets (.24g) displayed after being centrifuged, VDS
Figure 6: Day 3, 2/29/13, Fun Plate after two days of overnight incubation at 37 C, VDS
Don't need the second fun plate picture
Figure 7: Elution 1 represented as a light purple after the addition of 5ml of the wash buffer (.2ml imidazole) to remove loosely bound proteins
Figure 8: Elution 2 represented as a clear liquid after the addition of 5ml of the elution buffer (2.5ml imidazole) to release the gbr22 protein from the Ni-NTA resin
Figure 9. Run 1 of Elution 1 (2ul) using Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) at a wavelength of 280nm, absorbance recorded at 0.29 mg/ml.
Also show run 2 graph
Beer's Law Lab calculations A=Ebc A (Absorption): 0.030 b (cuvette path length): 1mm E (Extinction coefficient) at 280 nm: 33850/M*cm MW of gbr-22: 25794.2 g/mol
C=A/bE (0.30)/(38850/M*cm)= .1992 mg/mL (Add how you used the molecular weight to convert to mg/mL) Add maximal wavelength calculation
Yield at 280nm: (0.1992 mg/mL)(5mL)= 0.996 mg Using the absorbance reading and extinction coefficient at 574nm the yield for the maximal wavelength could also be determined using Beer's Law. Yield at Maximal Wavelength: (0.0806 mg/mL)(5mL)= 0.403 mg
Figure 9. Dry gel displaying cells 10-1 of gel electrophoresis results. Cells 1-6 representing cell solution, solution fraction, flow through, wash, elution 1 and elution 2. Cells 7-10 represent wash, elution 1, and elution 2 from the second partner's sample of gbr-22 protein purification samples.
I think you might have pasted in the wrong picture Gel used is a gradient of 4-20% that allows for the observation of a range of protein MW around 25 kDa.
Figure 10. Thermo Scientific Image of the molecule weight standard, blot and gel displayed in terms of kDa.
Discussion:
The lysozyme was used to break down the bacterial E.coli cell wall. This is important in order to ensure an efficient extraction of protein and obtain the purest sample possible. Cyanase (Benzonase) is a nuclease that digests the DNA/RNA from the solution, reducing its viscosity and farther isolating the protein. This enzyme is used to reduce the viscosity of the purple protein so that it can be administered through the syringe filter and into the transformation tube while removing larger particulate matter from the solution.
The elution buffer was added to the solution In order to purify the gbr-22 protein and remove other cellular proteins. The gbr-22 protein has approximately six histidine residues that are attached to the C-terminus. The protein can be separated from other cellular proteins through a series of cation and anion ion interactions. With the addition of Ni-NTA agarose the HIS tag will bind with the nickel cations. The Nickel cations are immobilized and release the protein with the addition of imidazole (Elution Buffer). The imidazole solution competes with the HIS tags in binding to the gbr-22 protein.
Sample 1 (cell solution) contains E.coli cells that are expressing the purple gbr-22 protein, the lysozyme enzyme, and cell wall fragments. Sample 2 (solution fraction) contains (soluble) bacterial proteins, gbr-22, and supernatant fluid. Sample 3 (flow through) contains proteins that did not adhere to the nickel; the gbr-22 protein was not collected in this sample because it was bound to the beads. Sample 4 (wash) contains loosely bound proteins. Sample 5 (Elution 1) contains a majority of gbr-22 protein. Sample 6 (Elution 2) contains extra remnants of gbr-22 protein.
The Wash buffer is used to remove loosely bound proteins and contains a weak amount of imidazole, while the elution buffer is used to remove tightly bound proteins and molecules from the gbr-22 protein. Specifically, the Elution buffer removes the gbr-22 protein from the Ni-NTA resin with a stronger concentration of imidazole than the wash buffer.
The size of the protein is 25 kDa from the gel which was the size determined in the protein purification lab.
There are some specific errors that could have occurred including environmental contaminantion, human miscalculations and contamination errors, and faulty equipment. Other possible errors could have occurred when obtaining purification samples of protein during purification. This is demonstrated in column 7-10 which appear very light. This suggests that not a lot of protein was obtained. This could have also occurred due to the lack of imidazole added to release the protein into sample 5. As seen in figure 9, a dark band appears below the gbr-22 protein band (25 kDa) at around 15 kDa. This is a contamination from another protein; this along with lighter bands indicates that the predicted purity of 80% is now an estimated 30%.
Conclusions: The protein gbr-22 was expressed in the host E.coli, purified through affinity chromatography and characterized by Gel Electrophoresis to determine the MW, yield and purity of the final product. Then data collected from UV-VIS spectroscopy measurements were used to estimate the overall protein concentration. In the gel analysis another protein was found to be present, indicating a reduced purity of the sample estimated at about 30%.
The gbr-22 protein could go through more trials of the production, purification and expression processes to yield a purer protein sample and overall yield. Other proteins could also be expressed in the wet lab and analyzed or alternative variables could be tested. Also, virtual drug screening could be applied with the GOLD program and PyMOL to screen for high scoring ligands that could then be tested in the wet lab.
References:
[1] Martin Hallberg, Protein Production and Purification, Nat Methods, 2008, 5, (2), 135-46.
[2] Timothy Tan, Zong Tan, Wei Tan, Gel electrophoresis, Biochemistry and Molecular Biology Education. 2007, 35, (5), 342-349.
Make intro more relevant to this lab
Introduction:
Lab strains of Escherichia coli bacteria serve as a sufficient expression host in recombinant protein production. This is because a wide variety of variables can be tested in a short amount of time and it is an inexpensive process [1]. The production of a protein with affinity tags, such as histidine, aid in protein purification. A histidine tag is small in size, rarely alters protein characteristics, and can be removed using metal affinity chromatography [1]. The lower-affinity contaminants will compete with the excess of histidine-tagged protein to farther purify the protein. To prepare the protein for these processes, enzymes that degrade nucleic acids and cell-wall material, such as Benzonase and lysozyme, should be administered to the protein solution. Imidazole can then be added to the cell extraction buffer to remove loosely bound proteins without removing large segments of the recombinant protein. Then, in an additional buffer elution step containing a stronger imidazole concentration, the protein can be removed and collected in the flow through to prepare for characterization. In protein characterization the molecular weight, purity, and yield of the protein will be analyzed through Gel electrophoresis. Gel electrophoresis requires the use of agarose gel, buffer solution, and dye [2]. In Gel Electrophoresis an electrical charge is applied to separate fragments based on their molecular weight and positive or negative migration. As the positively charges dyes migrate towards the cathode, the gel containing these dyes are placed in the wells of the tank so that the dyes will migrate through the gel towards the cathode [2].
Through a series of experimental procedures the recombinant protein, gbr-22, will be overexpressed in the bacterial host, E.coli. Gbr-22 will then be isolated and purified through affinity chromatography. The samples collected during the protein purification process will be used in Gel Electrophoresis to determine the estimated molecular weight, purity, and overall yield of the gbr-22 protein. The gbr-22 protein production and purification process will have some contamination present that will result in a protein purity of about 70%. This will later be demonstrated through gel analysis.
Materials & Methods:
In the protein expression process, two transformation tubes were centrifuged containing 25 microliters each of BL21(DE3) competent bacterial cells from NEB (put NEB in a citation & state). 1-2 microliters of plasmid (pGEM-gbr22) was administered into a transformation tube containing DNA; the other tube is the control (specify that control means no DNA). Heat shock tubes in 42 C waterbath for 45 seconds. Add SOC media to the incubator for 30 min at 37 C at 250 rpm and spread onto a plate to store overnight. Select a colony from the protein expression and transfer the cells into the LB/amp media to be placed in the shaking incubator. Grow at 37 C and 200-350 rpm. Centrifuge (Which centrifuge did you use) 500 ul (Centrifuged 25 mL, 500ul sample was taken before) of the sample for 10 min at 4 C and 5000 rpm. Add 1xPBS to the pellet, vortex, and add the enzyme lysozyme and store in a -4 C freezer.
For protein purification add 2 ul Benzonase, centrifuge for 20 min at 14,000 rpm and 4 C then run through 0.22 um filter (Which brand of filter). Add .5 ml of Ni-NTA buffer mix to the protein solution. Using a Econo column collect the flow through, leaving some of the purple pellet in fluid. Flow through the Wash buffer, 10ml of 1x PBS and 20mM imidazole, and twice with Elution buffer, 10mL of 1x PBS with 250 mM imidazole. Samples of cell solution, fraction, flow through, wash, Elu1, and Elu2 will be collected for use in gel electrophoresis. Use the Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE)/UV-VIS to measure the elution absorbance at 280nm and maximal wavelength.
In Protein characterization, add loading buffer to the samples collected in protein purification and administer into the cleared cell walls. Set the tank at 200 V for 25 min. Wash the gel and apply imperial protein stain. Cover gel with saran wrap and dry at 75 C for 1.5 hours on vacuum mode. (What type of gel did you use)
Results:
Beer's Law Lab calculations
A=Ebc
A (Absorption): 0.030
b (cuvette path length): 1mm
E (Extinction coefficient) at 280 nm: 33850/M*cm
MW of gbr-22: 25794.2 g/mol
C=A/bE
(0.30)/(38850/M*cm)= .1992 mg/mL (Add how you used the molecular weight to convert to mg/mL)
Add maximal wavelength calculation
Yield at 280nm: (0.1992 mg/mL)(5mL)= 0.996 mg
Using the absorbance reading and extinction coefficient at 574nm the yield for the maximal wavelength could also be determined using Beer's Law.
Yield at Maximal Wavelength: (0.0806 mg/mL)(5mL)= 0.403 mg
Gel used is a gradient of 4-20% that allows for the observation of a range of protein MW around 25 kDa.
The lysozyme was used to break down the bacterial E.coli cell wall. This is important in order to ensure an efficient extraction of protein and obtain the purest sample possible. Cyanase (Benzonase) is a nuclease that digests the DNA/RNA from the solution, reducing its viscosity and farther isolating the protein. This enzyme is used to reduce the viscosity of the purple protein so that it can be administered through the syringe filter and into the transformation tube while removing larger particulate matter from the solution.
The elution buffer was added to the solution In order to purify the gbr-22 protein and remove other cellular proteins. The gbr-22 protein has approximately six histidine residues that are attached to the C-terminus. The protein can be separated from other cellular proteins through a series of cation and anion ion interactions. With the addition of Ni-NTA agarose the HIS tag will bind with the nickel cations. The Nickel cations are immobilized and release the protein with the addition of imidazole (Elution Buffer). The imidazole solution competes with the HIS tags in binding to the gbr-22 protein.
Sample 1 (cell solution) contains E.coli cells that are expressing the purple gbr-22 protein, the lysozyme enzyme, and cell wall fragments. Sample 2 (solution fraction) contains (soluble) bacterial proteins, gbr-22, and supernatant fluid. Sample 3 (flow through) contains proteins that did not adhere to the nickel; the gbr-22 protein was not collected in this sample because it was bound to the beads. Sample 4 (wash) contains loosely bound proteins. Sample 5 (Elution 1) contains a majority of gbr-22 protein. Sample 6 (Elution 2) contains extra remnants of gbr-22 protein.
The Wash buffer is used to remove loosely bound proteins and contains a weak amount of imidazole, while the elution buffer is used to remove tightly bound proteins and molecules from the gbr-22 protein. Specifically, the Elution buffer removes the gbr-22 protein from the Ni-NTA resin with a stronger concentration of imidazole than the wash buffer.
The size of the protein is 25 kDa from the gel which was the size determined in the protein purification lab.
There are some specific errors that could have occurred including environmental contaminantion, human miscalculations and contamination errors, and faulty equipment. Other possible errors could have occurred when obtaining purification samples of protein during purification. This is demonstrated in column 7-10 which appear very light. This suggests that not a lot of protein was obtained. This could have also occurred due to the lack of imidazole added to release the protein into sample 5. As seen in figure 9, a dark band appears below the gbr-22 protein band (25 kDa) at around 15 kDa. This is a contamination from another protein; this along with lighter bands indicates that the predicted purity of 80% is now an estimated 30%.
Conclusions:
The protein gbr-22 was expressed in the host E.coli, purified through affinity chromatography and characterized by Gel Electrophoresis to determine the MW, yield and purity of the final product. Then data collected from UV-VIS spectroscopy measurements were used to estimate the overall protein concentration. In the gel analysis another protein was found to be present, indicating a reduced purity of the sample estimated at about 30%.
The gbr-22 protein could go through more trials of the production, purification and expression processes to yield a purer protein sample and overall yield. Other proteins could also be expressed in the wet lab and analyzed or alternative variables could be tested. Also, virtual drug screening could be applied with the GOLD program and PyMOL to screen for high scoring ligands that could then be tested in the wet lab.
References:
[1] Martin Hallberg, Protein Production and Purification, Nat Methods, 2008, 5, (2), 135-46.
[2] Timothy Tan, Zong Tan, Wei Tan, Gel electrophoresis, Biochemistry and Molecular Biology Education. 2007, 35, (5), 342-349.